Podosome regulated monocyte/macrophage tissue infiltration ABSTRACT While monocyte/macrophages (MDs) are essential components of innate immunity, they can also pose disadvantages to the host in certain pathological conditions such as chronic inflammatory diseases or cancer. It has been proposed that podosomes, F-actin rich ventral adhesion and protrusive structures, are employed by a range of leukocytes including MDs to not only mediate matrix degradation but to also play a role in MD chemotaxis and tissue recruitment. Wiskott-Aldrich syndrome protein (WASP), an activator of the actin nucleator Arp2/3 complex expressed exclusively in hematopoietic cells, is required for podosome formation and is critical for proper MD chemotaxis and matrix degradation. We have also recently identified non- redundant roles for WASP and the ubiquitously expressed family member N-WASP. This proposal will determine the precise role of WASP and N-WASP in chemotaxis, transendothelial migration (or diapedesis) and invasion. Previous work by us has demonstrated that tyrosine phosphorylation of WASP is a key factor in podosome turnover and chemotaxis and preliminary data suggest that the Src family tyrosine kinase Hck mediates WASP phosphorylation, which is required for MD diapedesis across an endothelium. In Specific Aim 1 we will determine the role of Intersectin 2L, a protein that only binds to phosphorylated WASP, in mediating WASP function. Intersectin 2L is a guanine nucleotide exchange factor for Cdc42. We hypothesize that WASP promotes the maintenance of Cdc42 activity by recruitment of Intersectin 2L during chemotaxis. A variety of techniques including siRNA, monitoring Cdc42 activity both in vitro and in vivo, and the creation of a photo- activatable form of Intersectin 2 will be utilized to test this hypothesis. The role of podosomes in chemotaxis, invasion and diapedesis will be explored in Specific Aim 2. We will determine the specific requirement of podosomes for chemotaxis using a variety of novel imaging approaches including the use of biosensors to analyze localized activity, photoconversion of podosome-associated actin to monitor kinetics and contributions of actin-rich compartments during chemotaxis, and photoablation of WASP or other components in podosomes by chromophore-assisted laser inactivation. In addition to WASP, we have demonstrated that N-WASP is also required for podosome-mediated matrix degradation and preliminary data suggest a requirement for WASP phosphorylation during transendothelial migration, potentially by affecting podosome dynamics at both the upper and lower surface of the endothelium. We will monitor the localization of podosomes and WASP activity during diapedesis using live cell imaging. The proteolytic function of podosomes during diapedesis and invasion will be assayed using an MMP biosensor and both pharmacological and genetic methods of inhibiting MMPs. Finally in Aim 3 we will determine the role of Hck, WASP and N-WASP in MD recruitment in vivo. MD diapedesis will be monitored using orthotopic tumors in Rag2-/- mice either with endogenous macrophages labeled with GFP in wild-type, Hck- or WASP-deficient mice or by tail vein injection of GFP labeled macrophages with down-regulation or expressing mutations in WASP, N-WASP or Intersectin2. In addition, we will use a new intravital imaging technique to obtain high resolution images of monocytes migrating into breast tumors in live mice. Characterizing the molecular events mediating MD chemotaxis and extravasation will lead to a better understanding of the recruitment of these cells to tumors. In addition, the results of this work may be applicable to chronic inflammatory diseases where MDs appear to play pathogenic roles, suggesting that WASP inhibition may have therapeutic benefits in the treatment of several different human diseases. PUBLIC HEALTH RELEVANCE: Project Narrative While monocyte/macrophages (MDs) are essential components of innate immunity;they can also pose disadvantages in certain pathological conditions such as chronic inflammatory diseases or cancer. Macrophages are therefore a prime target for therapies, but it is important to elucidate the mechanisms by which they are recruited to and activated in tissues. This project focuses on characterizing the molecular events mediating MD chemotaxis and extravasation that will lead to a better understanding of how these cells are recruited to tumors. In addition, the results of this work may be applicable to chronic inflammatory diseases where MDs appear to play pathogenic roles and aid in the identification of potential therapeutic targets in the treatment of several different human diseases.